title: Ultrahigh pressure fast size exclusion chromatography for top-down proteomics.
abstract: Top-down MS-based proteomics has gained a solid growth over the past few years but still faces significant challenges in the LC separation of intact proteins. In top-down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. SEC is a favored LC method for size-based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein, we reported the use of ultrahigh pressure (UHP) SEC for rapid and high-resolution separation of intact proteins for top-down proteomics. Fast separation of intact proteins (6-669 kDa) was achieved in < 7 min with high resolution and high efficiency. More importantly, we have shown that this UHP-SEC provides high-resolution separation of intact proteins using a MS-friendly volatile solvent system, allowing the direct top-down MS analysis of SEC-eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP-SEC is an attractive LC strategy for the size separation of proteins with great potential for top-down proteomics.
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, USA; Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
mesh_terms: D007611:Aprotinin / Q000032:analysis; D002850:Chromatography, Gel / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D045304:Cytochromes c / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods; D010047:Ovalbumin / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D013954:Thyroglobulin / Q000032:analysis
chemical_list: D007074:Immunoglobulin G; D011506:Proteins; D010047:Ovalbumin; D045304:Cytochromes c; D013954:Thyroglobulin; D007611:Aprotinin
keywords: Liquid chromatography; Mass spectrometry; Posttranslational modifications; Protein separation; Technology; Ultra performance liquid chromatography